SODD-specific antibodies and methods

ABSTRACT

The invention provides methods and compositions relating to Suppressor of Death Domain (SODD) proteins which regulate cellular signal transduction and transcriptional activation, and related nucleic acids. The polypeptides may be produced recombinantly from transformed host cells from the disclosed SODD encoding nucleic acids or purified from human cells. The invention provides isolated SODD hybridization probes and primers capable of specifically hybridizing with the disclosed SODD genes, SODD-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

This application is a divisional of U.S. Ser. No. 09/035,676, filed Mar.5, 1998.

FIELD OF THE INVENTION

The field of this invention is proteins involved in cell signaltransduction.

BACKGROUND

Tumor necrosis factor (TNF) is an important cytokine involved in thesignaling of a number of cellular responses including cytotoxicity,anti-viral activity, immun-regulatory activities and the transcriptionalregulation of a number of genes. The TNF receptors (TNFR1 and TNFR2) aremembers of the larger TNF receptor superfamily which also includes theFas antigen, CD27, CD30, CD40, and several other receptors (Smith etal., 1996, Cell 75, 959-962). Members of this family have been shown toparticipate in a variety of biological properties, including programmedcell death, antiviral activity and activation of the transcriptionfactor NF-κB in a wide variety of cell types. In particular, deathdomain containing members of this family, such as TNFR1 and Fas, caninduce programmed cell death through a shared ˜80 amino acid deathdomain (Tartaglia et al., 1993, Cell 74, 845-853; Itoh et al., 1993,J.Biol.Chem. 258, 10932-10937).

Additional intracellular death domain containing proteins are identifiedthrough yeast two-hybrid interaction cloning by virtue of theirinteractions with the death domains of death domain containing membersof the TNF receptor superfamily. For example, TRADD has been shown tointeract specifically with TNFR1 (Hsu, et al., 1995, Cell 81, 495-504)and FADD (Boldin et al., 1995, J.Biol.Chem. 270, 7795-7798; Chinnaiyanet al., 1995, Cell 81, 505-512) and RIP (Stanger et al., 1995, Cell 81,513-523) interact specifically with Fas. In fact, death domains defineinteraction domains that provide both homotypic and heterotypicassociations and can function as adapters to couple members of the TNFRsuperfamily with other signaling proteins (see, e.g. Hsu et al. (1996)Cell 84, 299-308).

Accordingly, the ability to exogenously modulate the activity of deathdomain containing proteins would yield therapeutic application fornumerous clinical indications. In addition, components of such pathwayswould provide valuable target reagents for automated, cost-effective,high throughput drug screening assays and hence would have immediateapplication in domestic and international pharmaceutical andbiotechnology drug development programs. The present invention providesnovel modulators of death domains, their use, e.g. in drug screens,modulating cellular function, etc.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to isolatedSuppressor of Death Domain (SODD) polypeptides, related nucleic acids,polypeptide domains thereof having SODD-specific structure and activityand modulators of SODD function, particularly death domain bindingactivity. SODD polypeptides can regulate death domain containingproteins, including members of the TNFR superfamily and hence provideimportant regulators of cell function such as NFκB activation. Thepolypeptides may be produced recombinantly from transformed host cellsfrom the subject SODD polypeptide encoding nucleic acids or purifiedfrom mammalian cells. The invention provides isolated SODD genehybridization probes and primers capable of specifically hybridizingwith the disclosed SODD-encoding genes, SODD-specific binding agentssuch as specific antibodies, and methods of making and using the subjectcompositions in diagnosis (e.g. nucleic acid hybridization screens forSODD transcripts), modulating cellular physiology (e.g. modulatingintracellular SODD activity to modulate TNF signal transduction) and inthe biopharmaceutical industry (e.g. as immunogens, reagents forisolating other transcriptional regulators, reagents for screeningchemical libraries for lead pharmacological agents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

The nucleotide sequence of a natural cDNA encoding a human SODDpolypeptide is shown as SEQ ID NO:1, and the full conceptual translateis shown as SEQ ID NO:2. The SODD polypeptides of the invention includeone or more functional domains of SEQ ID NO:2, which domains comprise atleast 8, preferably at least 16, more preferably at least 32, mostpreferably at least 64 contiguous residues of SEQ ID NO:2 including atleast one, preferably at least two, more preferably at least 4 and mostpreferably all of said contiguous residues contained within at least oneof residues 1-122, residues 180-237 and residues 450-457, and have humanSODD-specific amino acid sequence and activity. SODD domain specificactivities include TNFR superfamily death domain-binding and/or bindinginhibitory activity and SODD-specific immunogenicity and/orantigenicity.

SODD-specific activity or function may be determined by convenient invitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cellculture assays, in animals (e.g. gene therapy, transgenics, etc.), etc.Binding assays encompass any assay where the molecular interaction of anSODD polypeptide with a binding target is evaluated. The binding targetmay be a natural intracellular binding target such as an SODD bindingtarget, a SODD regulating protein or other regulator that directlymodulates SODD activity or its localization; or non-natural bindingtarget such a specific immune protein such as an antibody, or an SODDspecific agent such as those identified in screening assays such asdescribed below. SODD-binding specificity may be assayed by bindingequilibrium constants (usually at least about 10⁷ M⁻¹, preferably atleast about 10⁸ M⁻¹, more preferably at least about 10⁹ M⁻¹), by NFκBreporter expression, by apoptosis assays, by the ability of the subjectpolypeptide to function as negative mutants in SODD-expressing cells, toelicit SODD specific antibody in a heterologous host (e.g a rodent orrabbit), etc.

For example, deletion mutagenesis is used to defined functional SODDdomains which bind and sequester TNFR superfamily death domains, inhibitapoptosis or inhibit NFκB expression in SODD-modulated NFκB activationassays. See, e.g. Table 1.

                  TABLE 1                                                         ______________________________________                                        Exemplary SODD deletion mutants defining SODD functional domains.                                    Death                                                  Mu-                    Domain  Apoptosis                                                                            NFκB                              tant Sequence          Binding Inhibition                                                                           Inhibition                              ______________________________________                                        ΔN1                                                                          SEQ ID NO:2, residues 23-457                                                                    +       +      +                                       ΔN2                                                                          SEQ ID NO:2, residues 68-457                                                                    +       +      +                                       ΔN3                                                                          SEQ ID NO:2, residues 118-457                                                                   +       +      +                                       ΔN4                                                                          SEQ ID NO:2, residues 185-457                                                                   +       +      +                                       ΔN5                                                                          SEQ ID NO:2, residues 261-457                                                                   +       +      +                                       ΔC1                                                                          SEQ ID NO:2, residues 1-456                                                                     +       +      +                                       ΔC2                                                                          SEQ ID NO:2, residues 1-420                                                                     -       -      -                                       ΔC3                                                                          SEQ ID NO:2, residues 1-373                                                                     -       -      -                                       ΔC4                                                                          SEQ ID NO:2, residues 1-277                                                                     -       -      -                                       ______________________________________                                    

In a particular embodiment, the subject domains provide SODD-specificantigens and/or immunogens, especially when coupled to carrier proteins.For example, peptides corresponding to SODD- and human SODD-specificdomains are covalently coupled to keyhole limpet antigen (KLH) and theconjugate is emulsified in Freunds complete adjuvant. Laboratory rabbitsare immunized according to conventional protocol and bled. The presenceof SODD-specific antibodies is assayed by solid phase immunosorbantassays using immobilized SODD polypeptides of SEQ ID NO:2, see, e.g.Table 2.

                  TABLE 2                                                         ______________________________________                                        Immunogenic SODD polypeptides eliciting SODD-specific rabbit                  polyclonal antibody: SODD polypeptide-KLH conjugates immunized                per protocol described above.                                                 SODD Polypeptide Sequence                                                                        Immunogenicity                                             ______________________________________                                        SEQ ID NO:2, residues 1-10                                                                       +++                                                        SEQ ID NO:2, residues 12-21                                                                      +++                                                        SEQ ID NO:2, residues 25-37                                                                      +++                                                        SEQ ID NO:2, residues 42-59                                                                      +++                                                        SEQ ID NO:2, residues 62-71                                                                      +++                                                        SEQ ID NO:2, residues 72-85                                                                      +++                                                        SEQ ID NO:2, residues 88-89                                                                      +++                                                        SEQ ID NO:2, residues 105-112                                                                    +++                                                        SEQ ID NO:2, residues 116-122                                                                    +++                                                        SEQ ID NO:2, residues 120-128                                                                    +++                                                        SEQ ID NO:2, residues 175-182                                                                    +++                                                        SEQ ID NO:2, residues 180-195                                                                    +++                                                        SEQ ID NO:2, residues 201-208                                                                    +++                                                        SEQ ID NO:2, residues 213-222                                                                    +++                                                        SEQ ID NO:2, residues 222-230                                                                    +++                                                        SEQ ID NO:2, residues 228-237                                                                    +++                                                        SEQ ID NO:2, residues 230-338                                                                    +++                                                        SEQ ID NO:2, residues 237-245                                                                    +++                                                        SEQ ID NO:2, residues 440-450                                                                    +++                                                        SEQ ID NO:2, residues 442-451                                                                    +++                                                        SEQ ID NO:2, residues 445-452                                                                    +++                                                        SEQ ID NO:2, residues 447-454                                                                    +++                                                        SEQ ID NO:2, residues 449-456                                                                    +++                                                        SEQ ID NO:2, residues 450-457                                                                    +++                                                        ______________________________________                                    

The claimed SODD polypeptides are isolated or pure: an "isolated"polypeptide is unaccompanied by at least some of the material with whichit is associated in its natural state, preferably constituting at leastabout 0.5%, and more preferably at least about 5% by weight of the totalpolypeptide in a given sample and a pure polypeptide constitutes atleast about 90%, and preferably at least about 99% by weight of thetotal polypeptide in a given sample. The SODD polypeptides andpolypeptide domains may be synthesized, produced by recombinanttechnology, or purified from mammalian, preferably human cells. A widevariety of molecular and biochemical methods are available forbiochemical synthesis, molecular expression and purification of thesubject compositions, see e.g. Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides binding agents specific to SODD polypeptides,preferably the claimed SODD polypeptides, including substrates,agonists, antagonists, natural intracellular binding targets, etc.,methods of identifying and making such agents, and their use indiagnosis, therapy and pharmaceutical development. For example, specificbinding agents are useful in a variety of diagnostic and therapeuticapplications, especially where disease or disease prognosis isassociated with improper utilization of a pathway involving the subjectproteins, e.g TNF signal transduction. Novel SODD-specific bindingagents include SODD-specific receptors, such as somatically recombinedpolypeptide receptors like specific antibodies or T-cell antigenreceptors (see, e.g Harlow and Lane (1988) Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory) and other natural intracellularbinding agents identified with assays such as one-, two- andthree-hybrid screens, non-natural intracellular binding agentsidentified in screens of chemical libraries such as described below,etc. Agents of particular interest modulate SODD function, e.g.SODD-modulatable signal transduction.

Accordingly, the invention provides methods for modulating apoptosisand/or death domain function, particularly TNFR superfamily death domainfunction, more particularly, TNFR1 and/or DR3 death domain function in acell comprising the step of modulating SODD activity. The cell mayreside in culture or in situ, i.e. within the natural host. Generally,agents are introduced into the cell which effect an upregulation in theactivity of intracellular SODD polypeptides. For example, SODD-encodingpolypeptides may be transfected into the cell under conditions whicheffect the expression of the encoded SODD polypeptides.

The amino acid sequences of the disclosed SODD polypeptides are used toback-translate SODD polypeptide-encoding nucleic acids optimized forselected expression systems (Holler et al. (1993) Gene 136, 323-328;Martin et al. (1995) Gene 154, 150-166) or used to generate degenerateoligonucleotide primers and probes for use in the isolation of naturalSODD-encoding nucleic acid sequences ("GCG" software, Genetics ComputerGroup, Inc, Madison Wis.). SODD-encoding nucleic acids used inSODD-expression vectors and incorporated into recombinant host cells,e.g. for expression and screening, transgenic animals, e.g. forfunctional studies such as the efficacy of candidate drugs for diseaseassociated with SODD-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a SODD cDNA specific sequencecomprising at least 12, preferably at least 24, more preferably at least36 and most preferably at least contiguous 96 bases of a strand of SEQID NO:1 including at least two contiguous nucleotides contained withinat least one of nucleotides 1-366, nucleotides 537-711 and residues1348-1444 and preferably sufficient to specifically hybridize with asecond nucleic acid comprising the complementary strand of SEQ ID NO:1.Demonstrating specific hybridization generally requires stringentconditions, for example, hybridizing in a buffer comprising 30%formainide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH7.7, 0.001 M EDTA)buffer at a temperature of 42° C. and remaining bound when subject towashing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffercomprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. andremaining bound when subject to washing at 42° C. with 0.2×SSPE bufferat 42° C.

                  TABLE 3                                                         ______________________________________                                        Exemplary SODD nucleic acids which hybridize with a strand of                 SEQ ID NO:1 under Conditions I and/or II.                                     SODD Nucleic Acids   Hybridization                                            ______________________________________                                        SEQ ID NO:1, nucleotides 1-36                                                                      +                                                        SEQ ID NO:1, nucleotides 68-98                                                                     +                                                        SEQ ID NO:1, nucleotides 95-130                                                                    +                                                        SEQ ID NO:1, nucleotides 175-220                                                                   +                                                        SEQ ID NO:1, nucleotides 261-299                                                                   +                                                        SEQ ID NO:1, nucleotides 274-310                                                                   +                                                        SEQ ID NO:1, nucleotides 331-369                                                                   +                                                        SEQ ID NO:1, nucleotides 530-570                                                                   +                                                        SEQ ID NO:1, nucleotides 584-616                                                                   +                                                        SEQ ID NO:1, nucleotides 661-708                                                                   +                                                        SEQ ID NO:1, nucleotides 689-725                                                                   +                                                        SEQ ID NO:1, nucleotides 1328-1365                                                                 +                                                        SEQ ID NO:1, nucleotides 1338-1358                                                                 +                                                        SEQ ID NO:1, nucleotides 1348-1372                                                                 +                                                        SEQ ID NO:1, nucleotides 1365-1399                                                                 +                                                        SEQ ID NO:1, nucleotides 1402-1423                                                                 +                                                        SEQ ID NO:1, nucleotides 1417-1444                                                                 +                                                        ______________________________________                                    

The subject nucleic acids are of synthetic/non-natural sequences and/orare isolated, i.e. unaccompanied by at least some of the material withwhich it is associated in its natural state, preferably constituting atleast about 0.5%, preferably at least about 5% by weight of totalnucleic acid present in a given fraction, and usually recombinant,meaning they comprise a non-natural sequence or a natural sequencejoined to nucleotide(s) other than that which it is joined to on anatural chromosome. Recombinant nucleic acids comprising the nucleotidesequence of SEQ ID NO:1, or requisite fragments thereof, contain suchsequence or fragment at a terminus, immediately flanked by (i.e.contiguous with) a sequence other than that which it is joined to on anatural chromosome, or flanked by a native flanking region fewer than 10kb, preferably fewer than 2 kb, which is at a terminus or is immediatelyflanked by a sequence other than that which it is joined to on a naturalchromosome. While the nucleic acids are usually RNA or DNA, it is oftenadvantageous to use nucleic acids comprising other bases or nucleotideanalogs to provide modified stability, etc.

The subject nucleic acids find a wide variety of applications includinguse as translatable transcripts, hybridization probes, PCR primers,diagnostic nucleic acids, etc.; use in detecting the presence of SODDgenes and gene transcripts and in detecting or amplifying nucleic acidsencoding additional SODD homologs and structural analogs. In diagnosis,SODD hybridization probes find use in identifying wild-type and mutantSODD alleles in clinical and laboratory samples. Mutant alleles are usedto generate allele-specific oligonucleotide (ASO) probes forhigh-throughput clinical diagnoses. In therapy, therapeutic SODD nucleicacids are used to modulate cellular expression or intracellularconcentration or availability of active SODD.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of a SODDmodulatable cellular function. Generally, these screening methodsinvolve assaying for compounds which modulate SODD interaction with anatural SODD binding target. A wide variety of assays for binding agentsare provided including labeled in vitro protein-protein binding assays,immunoassays, cell based assays, etc. The methods are amenable toautomated, cost-effective high throughput screening of chemicallibraries for lead compounds. Identified reagents find use in thepharmaceutical industries for animal and human trials; for example, thereagents may be derivatized and rescreened in in vitro and in vivoassays to optimize activity and minimize toxicity for pharmaceuticaldevelopment.

In vitro binding assays employ a mixture of components including an SODDpolypeptide, which may be part of a fusion product with another peptideor polypeptide, e.g. a tag for detection or anchoring, etc. The assaymixtures comprise a natural intracellular SODD binding target. Whilenative full-length binding targets may be used, it is frequentlypreferred to use portions (e.g. peptides) thereof so long as the portionprovides binding affinity and avidity to the subject SODD polypeptideconveniently measurable in the assay. The assay mixture also comprises acandidate pharmacological agent. Candidate agents encompass numerouschemical classes, though typically they are organic compounds;preferably small organic compounds and are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Avariety of other reagents may also be included in the mixture. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, protease inhibitors, nuclease inhibitors, antimicrobialagents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for thepresence of the candidate pharmacological agent, the SODD polypeptidespecifically binds the cellular binding target, portion or analog with areference binding affinity. The mixture components can be added in anyorder that provides for the requisite bindings and incubations may beperformed at any temperature which facilitates optimal binding.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the SODD polypeptideand one or more binding targets is detected by any convenient way. Adifference in the binding affinity of the SODD polypeptide to the targetin the absence of the agent as compared with the binding affinity in thepresence of the agent indicates that the agent modulates the binding ofthe SODD polypeptide to the SODD binding target. Analogously, in thecell-based assay also described below, a difference in SODD-dependenttranscriptional activation in the presence and absence of an agentindicates the agent modulates SODD function. A difference, as usedherein, is statistically significant and preferably represents at leasta 50%, more preferably at least a 90% difference.

The following experimental section and examples are offered by way ofillustration and not by way of limitation.

EXAMPLES

1. Experimental Procedures

Recombinant human TNF and MAb 985 against the extracellular domain ofTNFR1 were obtained from Genentech, Inc.; anti-Flag MAb M2 from EastmanKodak Company; and anti-Flag polyclonal antibodies from Santa CruzBiotechnology. SODD polypeptide specific polyclonal antisera weregenerated against a SODD peptide (residues 292 to 313) and againstHis-tagged native SODD protein. Human embryonic kidney 293 cells, HeLacells, U937 cells and Jurkat cells were maintained as described by Hsuet al. (1996) Cell 84, 299-308.

A native human SODD protein was originally identified as aDR3-interacting protein by two hybrid screening. The plasmid Gal4BD-DR3,which encodes the GAL4 DNA-binding domain fused to the intracellulardomain of DR3, was used as bait in two-hybrid screens of a HeLa cellcDNA library (Clontech). The isolated positive clones were analyzed asdescribed by Hsu et al. (supra). Full length SODD cDNA was obtained byscreening a human Jurkat T cell cDNA library in lZAP by standard methods(Sambrook et al., 1989).

For northern analysis, the native SODD cDNA was used to probed a humanmultiple tissue Northern blot (Clontech), according to themanufacturer's protocol. These assays revealed broad tissue distributionof SODD transcripts, including immune cells.

Mammalian cell expression vectors encoding TNFR1, TRADD, crmA andcontrol plasmid pRK5 have been described previously Hsu et al. (supra).An expression vector for N-terminally Flag-tagged SODD polypeptide wasconstructed in the expression vector in pRK. Deletion mutants of nativeSODD protein were generated by inserting PCR fragments encodingcorresponding SODD amino acid sequences in-frame with an N-terminal Flagepitope coding sequence in the vector pRK.

For coimmunoprecipitation assays, subconfluent 10 cm dish cultures of293 cells were transfected by the calcium phosphate method. 24 hoursafter transfection, cells were washed in PBS and lysed in EIA lysisbuffer {Hsu}. Lysates were incubated for 2-4 hours at 4° C. withanti-TNFR1 monoclonal antibody (985) or control mouse IgG monoclonalantibody (Sigma) and 25 ml of 1:1 slurry of protein G-Sepharose(Pharmacia). Beads were washed twice with 1 ml E1A buffer, twice with 1ml of high salt (1 M NaCl) E1A buffer, and twice again with E1A buffer.The precipitates were fractionated on SDS-PAGE and transferred tonitrocellulose membrane. Western blotting analyzes were performed usingstandard procedures.

For endogenous association assay, U937 cells (2×10⁸) and Jurkat cells(5×10⁷) were washed with warm PBS, and incubated at 15 min in thepresence or absence of TNF (100 ng/ml). Cells were lysed in Triton-X-100lysis buffer and resultant lysates incubated with 25 mg of MAb 985 ormouse IgG and protein G-Sepharose (Pharmacia) at 4° C. overnight. Thebeads were washed twice with lysis buffer, twice with 1 ml high saltlysis buffer, and again with lysis buffer. The samples were separated onSDS-PAGE, and analyzed as above.

2. Protocol for Cell-Based NF-κB Reporter Assay

SODD can inhibit transactivation of NF-κB reporter constructs whenoverexpressed in 293 cells or HeLa cells. 293 cells are transfectedusing the calcium phosphate precipitation method with a plasmid encodinga 6 NF-κB-luciferase reporter construct and various amounts ofexpression vector encoding SODD. After 36-48 hours, cells are leftuntreated or treated with IL-1 (10-50 ng/ml) or TNF (50-100 ng) for 6hours prior to harvest. Cells are lysed and luciferase activity measuredusing the luciferase assay kit (Promega). The luciferase activity ineach transfection is normalized by co-transfecting a pRSV-β gal controlvector.

3. Protocol for High Throughput in vitro SODD-TNFR1 Death Domain BindingAssay

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol,0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of proteaseinhibitors.

³³ P SODD polypeptide 10× stock: 10⁻⁸ -10⁻⁶ M "cold" SODD supplementedwith 200,000-250,000 cpm of labeled SODD (Beckman counter). Place in the4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575),and 2 mM NaVO₃ (Sigma # S-6508) in 10 ml of PBS.

TNFR1 deletion mutant: 10⁻⁷ -10⁻⁵ M biotinylated TNFR1 80 residue deathdomain in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³ P-SODD (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹ -10⁻⁷ M finalconc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated TNFR1 deletion mutant (0.1-10 pmoles/40 ul inassay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated TNFR1 deletion mutant) at 80% inhibition.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1444 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1371                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATG TCG GCC CTG AGG CGC TCG GGC TAC GGC CC - #C AGT GAC GGT CCG TCC           48                                                                          Met Ser Ala Leu Arg Arg Ser Gly Tyr Gly Pr - #o Ser Asp Gly Pro Ser           #                 15                                                          - TAC GGC CGC TAC TAC GGG CCT GGG GGT GGA GA - #T GTG CCG GTA CAC CCA           96                                                                          Tyr Gly Arg Tyr Tyr Gly Pro Gly Gly Gly As - #p Val Pro Val His Pro           #             30                                                              - CCT CCA CCC TTA TAT CCT CTT CGC CCT GAA CC - #T CCC CAG CCT CCC ATT          144                                                                          Pro Pro Pro Leu Tyr Pro Leu Arg Pro Glu Pr - #o Pro Gln Pro Pro Ile           #         45                                                                  - TCC TGG CGG GTG CGC GGG GGC GGC CCG GCG GA - #G ACC ACC TGG CTG GGA          192                                                                          Ser Trp Arg Val Arg Gly Gly Gly Pro Ala Gl - #u Thr Thr Trp Leu Gly           #     60                                                                      - GAA GGC GGA GGA GGC GAT GGC TAC TAT CCC TC - #G GGA GGC GCC TGG CCA          240                                                                          Glu Gly Gly Gly Gly Asp Gly Tyr Tyr Pro Se - #r Gly Gly Ala Trp Pro           # 80                                                                          - GAG CCT GGT CGA GCC GGA GGA AGC CAC CAG GA - #G CAG CCA CCA TAT CCT          288                                                                          Glu Pro Gly Arg Ala Gly Gly Ser His Gln Gl - #u Gln Pro Pro Tyr Pro           #                 95                                                          - AGC TAC AAT TCT AAC TAT TGG AAT TCT ACT GC - #G AGA TCT AGG GCT CCT          336                                                                          Ser Tyr Asn Ser Asn Tyr Trp Asn Ser Thr Al - #a Arg Ser Arg Ala Pro           #           110                                                               - TAC CCA AGT ACA TAT CCT GTA AGA CCA GAA TT - #G CAA GGC CAG AGT TTG          384                                                                          Tyr Pro Ser Thr Tyr Pro Val Arg Pro Glu Le - #u Gln Gly Gln Ser Leu           #       125                                                                   - AAT TCT TAT ACA AAT GGA GCG TAT GGT CCA AC - #A TAC CCC CCA GGC CCT          432                                                                          Asn Ser Tyr Thr Asn Gly Ala Tyr Gly Pro Th - #r Tyr Pro Pro Gly Pro           #   140                                                                       - GGG GCA AAT ACT GCC TCA TAC TCA GGG GCT TA - #T TAT GCA CCT GGT TAT          480                                                                          Gly Ala Asn Thr Ala Ser Tyr Ser Gly Ala Ty - #r Tyr Ala Pro Gly Tyr           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - ACT CAG ACC AGT TAC TCC ACA GAA GTT CCA AG - #T ACT TAC CGT TCA TCT          528                                                                          Thr Gln Thr Ser Tyr Ser Thr Glu Val Pro Se - #r Thr Tyr Arg Ser Ser           #               175                                                           - GGC AAC AGC CCA ACT CCA GTC TCT CGT TGG AT - #C TAT CCC CAG CAG GAC          576                                                                          Gly Asn Ser Pro Thr Pro Val Ser Arg Trp Il - #e Tyr Pro Gln Gln Asp           #           190                                                               - TGT CAG ACT GAA GCA CCC CCT CTT AGG GGG CA - #G GTT CCA GGA TAT CCG          624                                                                          Cys Gln Thr Glu Ala Pro Pro Leu Arg Gly Gl - #n Val Pro Gly Tyr Pro           #       205                                                                   - CCT TCA CAG AAC CCT GGA ATG ACC CTG CCC CA - #T TAT CCT TAT GGA GAT          672                                                                          Pro Ser Gln Asn Pro Gly Met Thr Leu Pro Hi - #s Tyr Pro Tyr Gly Asp           #   220                                                                       - GGT AAT CGT AGT GTT CCA CAA TCA GGA CCG AC - #T GTA CGA CCA CAA GAA          720                                                                          Gly Asn Arg Ser Val Pro Gln Ser Gly Pro Th - #r Val Arg Pro Gln Glu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GAT GCG TGG GCT TCT CCT GGT GCT TAT GGA AT - #G GGT GGC CGT TAT CCC          768                                                                          Asp Ala Trp Ala Ser Pro Gly Ala Tyr Gly Me - #t Gly Gly Arg Tyr Pro           #               255                                                           - TGG CCT TCA TCA GCG CCC TCA GCA CCA CCC GG - #C AAT CTC TAC ATG ACT          816                                                                          Trp Pro Ser Ser Ala Pro Ser Ala Pro Pro Gl - #y Asn Leu Tyr Met Thr           #           270                                                               - GAA AGT ACT TCA CCA TGG CCT AGC AGT GGC TC - #T CCC CAG TCA CCC CCT          864                                                                          Glu Ser Thr Ser Pro Trp Pro Ser Ser Gly Se - #r Pro Gln Ser Pro Pro           #       285                                                                   - TCA CCC CCA GTC CAG CAG CCC AAG GAT TCT TC - #A TAC CCC TAT AGC CAA          912                                                                          Ser Pro Pro Val Gln Gln Pro Lys Asp Ser Se - #r Tyr Pro Tyr Ser Gln           #   300                                                                       - TCA GAT CAA AGC ATG AAC CGG CAC AAC TTT CC - #T TGC AGT GTC CAT CAG          960                                                                          Ser Asp Gln Ser Met Asn Arg His Asn Phe Pr - #o Cys Ser Val His Gln           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - TAC GAA TCC TCG GGG ACA GTG AAC AAT GAT GA - #T TCA GAT CTT TTG GAT         1008                                                                          Tyr Glu Ser Ser Gly Thr Val Asn Asn Asp As - #p Ser Asp Leu Leu Asp           #               335                                                           - TCC CAA GTC CAG TAT AGT GCT GAG CCT CAG CT - #G TAT GGT AAT GCC ACC         1056                                                                          Ser Gln Val Gln Tyr Ser Ala Glu Pro Gln Le - #u Tyr Gly Asn Ala Thr           #           350                                                               - AGT GAC CAT CCC AAC AAT CAA GAT CAA AGT AG - #C AGT CTT CCT GAA GAA         1104                                                                          Ser Asp His Pro Asn Asn Gln Asp Gln Ser Se - #r Ser Leu Pro Glu Glu           #       365                                                                   - TGT GTA CCT TCA GAT GAA AGT ACT CCT CCG AG - #T ATT AAA AAA ATC ATA         1152                                                                          Cys Val Pro Ser Asp Glu Ser Thr Pro Pro Se - #r Ile Lys Lys Ile Ile           #   380                                                                       - CAT GTG CTG GAG AAG GTC CAG TAT CTT GAA CA - #A GAA GTA GAA GAA TTT         1200                                                                          His Val Leu Glu Lys Val Gln Tyr Leu Glu Gl - #n Glu Val Glu Glu Phe           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - GTA GGA AAA AAG ACA GAC AAA GCA TAC TGG CT - #T CTG GAA GAA ATG CTA         1248                                                                          Val Gly Lys Lys Thr Asp Lys Ala Tyr Trp Le - #u Leu Glu Glu Met Leu           #               415                                                           - ACC AAG GAA CTT TTG GAA CTG GAT TCA GTT GA - #A ACT GGG GGC CAG GAC         1296                                                                          Thr Lys Glu Leu Leu Glu Leu Asp Ser Val Gl - #u Thr Gly Gly Gln Asp           #           430                                                               - TCT GTA CGG CAG GCC AGA AAA GAG GCT GTT TG - #T AAG ATT CAG GCC ATA         1344                                                                          Ser Val Arg Gln Ala Arg Lys Glu Ala Val Cy - #s Lys Ile Gln Ala Ile           #       445                                                                   - CTG GAA AAA TTA GAA AAA AAA GGA TTA TGAAAGGAT - #T TAGAACAAAG               1391                                                                          Leu Glu Lys Leu Glu Lys Lys Gly Leu                                           #   455                                                                       - GTCGACGCGG CCGCGAATTC CAGATCTATG AATCGTAGAT ACTGAAAAAC CC - #C              1444                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 457 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ser Ala Leu Arg Arg Ser Gly Tyr Gly Pr - #o Ser Asp Gly Pro Ser         #                 15                                                          - Tyr Gly Arg Tyr Tyr Gly Pro Gly Gly Gly As - #p Val Pro Val His Pro         #             30                                                              - Pro Pro Pro Leu Tyr Pro Leu Arg Pro Glu Pr - #o Pro Gln Pro Pro Ile         #         45                                                                  - Ser Trp Arg Val Arg Gly Gly Gly Pro Ala Gl - #u Thr Thr Trp Leu Gly         #     60                                                                      - Glu Gly Gly Gly Gly Asp Gly Tyr Tyr Pro Se - #r Gly Gly Ala Trp Pro         # 80                                                                          - Glu Pro Gly Arg Ala Gly Gly Ser His Gln Gl - #u Gln Pro Pro Tyr Pro         #                 95                                                          - Ser Tyr Asn Ser Asn Tyr Trp Asn Ser Thr Al - #a Arg Ser Arg Ala Pro         #           110                                                               - Tyr Pro Ser Thr Tyr Pro Val Arg Pro Glu Le - #u Gln Gly Gln Ser Leu         #       125                                                                   - Asn Ser Tyr Thr Asn Gly Ala Tyr Gly Pro Th - #r Tyr Pro Pro Gly Pro         #   140                                                                       - Gly Ala Asn Thr Ala Ser Tyr Ser Gly Ala Ty - #r Tyr Ala Pro Gly Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Gln Thr Ser Tyr Ser Thr Glu Val Pro Se - #r Thr Tyr Arg Ser Ser         #               175                                                           - Gly Asn Ser Pro Thr Pro Val Ser Arg Trp Il - #e Tyr Pro Gln Gln Asp         #           190                                                               - Cys Gln Thr Glu Ala Pro Pro Leu Arg Gly Gl - #n Val Pro Gly Tyr Pro         #       205                                                                   - Pro Ser Gln Asn Pro Gly Met Thr Leu Pro Hi - #s Tyr Pro Tyr Gly Asp         #   220                                                                       - Gly Asn Arg Ser Val Pro Gln Ser Gly Pro Th - #r Val Arg Pro Gln Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Ala Trp Ala Ser Pro Gly Ala Tyr Gly Me - #t Gly Gly Arg Tyr Pro         #               255                                                           - Trp Pro Ser Ser Ala Pro Ser Ala Pro Pro Gl - #y Asn Leu Tyr Met Thr         #           270                                                               - Glu Ser Thr Ser Pro Trp Pro Ser Ser Gly Se - #r Pro Gln Ser Pro Pro         #       285                                                                   - Ser Pro Pro Val Gln Gln Pro Lys Asp Ser Se - #r Tyr Pro Tyr Ser Gln         #   300                                                                       - Ser Asp Gln Ser Met Asn Arg His Asn Phe Pr - #o Cys Ser Val His Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Tyr Glu Ser Ser Gly Thr Val Asn Asn Asp As - #p Ser Asp Leu Leu Asp         #               335                                                           - Ser Gln Val Gln Tyr Ser Ala Glu Pro Gln Le - #u Tyr Gly Asn Ala Thr         #           350                                                               - Ser Asp His Pro Asn Asn Gln Asp Gln Ser Se - #r Ser Leu Pro Glu Glu         #       365                                                                   - Cys Val Pro Ser Asp Glu Ser Thr Pro Pro Se - #r Ile Lys Lys Ile Ile         #   380                                                                       - His Val Leu Glu Lys Val Gln Tyr Leu Glu Gl - #n Glu Val Glu Glu Phe         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Gly Lys Lys Thr Asp Lys Ala Tyr Trp Le - #u Leu Glu Glu Met Leu         #               415                                                           - Thr Lys Glu Leu Leu Glu Leu Asp Ser Val Gl - #u Thr Gly Gly Gln Asp         #           430                                                               - Ser Val Arg Gln Ala Arg Lys Glu Ala Val Cy - #s Lys Ile Gln Ala Ile         #       445                                                                   - Leu Glu Lys Leu Glu Lys Lys Gly Leu                                         #   455                                                                       __________________________________________________________________________

What is claimed is:
 1. An isolated antibody which specifically binds aSuppressor of Death Domain (SODD) polypeptide consisting of a sequenceselected from the group consisting of residues 25-37, 88-89, 105-112,175-182, 201-208, 222-230, 228-237, 237-245, 440-450 and 442-451 of SEQID NO:2.
 2. An antibody according to claim 1, wherein the SODDpolypeptide sequence consists of residues 25-37 of SEQ ID NO:2.
 3. Anantibody according to claim 1, wherein the SODD polypeptide sequenceconsists of residues 88-89 of SEQ ID NO:2.
 4. An antibody according toclaim 1, wherein the SODD polypeptide sequence consists of residues105-112 of SEQ ID NO:2.
 5. An antibody according to claim 1, wherein theSODD polypeptide sequence consists of residues 175-182 of SEQ ID NO:2.6. An antibody according to claim 1, wherein the SODD polypeptidesequence consists of residues 201-208 of SEQ ID NO:2.
 7. An antibodyaccording to claim 1, wherein the SODD polypeptide sequence consists ofresidues 222-230 of SEQ ID NO:2.
 8. An antibody according to claim 1,wherein the SODD polypeptide sequence consists of residues 228-237 ofSEQ ID NO:2.
 9. An antibody according to claim 1, wherein the SODDpolypeptide sequence consists of residues 237-245 of SEQ ID NO:2.
 10. Anantibody according to claim 1, wherein the SODD polypeptide sequenceconsists of residues 440-450 of SEQ ID NO:2.
 11. An antibody accordingto claim 1, wherein the SODD polypeptide sequence consists of residues442-451 of SEQ ID NO:2.
 12. A method for detecting a SODD polypeptide ina sample comprising the steps of:(a) contacting a sample with anantibody according to claim 1, and (b) detecting specific binding of theantibody to the SODD polypeptide as an indication of the presence of theSODD polypeptide in the sample.
 13. A method for detecting a SODDpolypeptide in a sample comprising the steps of:(a) contacting a samplewith an antibody according to claim 2, and (b) detecting specificbinding of the antibody to the SODD polypeptide as an indication of thepresence of the SODD polypeptide in the sample.
 14. A method fordetecting a SODD polypeptide in a sample comprising the steps of:(a)contacting a sample with an antibody according to claim 3, and (b)detecting specific binding of the antibody to the SODD polypeptide as anindication of the presence of the SODD polypeptide in the sample.
 15. Amethod for detecting a SODD polypeptide in a sample comprising the stepsof:(a) contacting a sample with an antibody according to claim 4, and(b) detecting specific binding of the antibody to the SODD polypeptideas an indication of the presence of the SODD polypeptide in the sample.16. A method for detecting a SODD polypeptide in a sample comprising thesteps of:(a) contacting a sample with an antibody according to claim 5,and (b) detecting specific binding of the antibody to the SODDpolypeptide as an indication of the presence of the SODD polypeptide inthe sample.
 17. A method for detecting a SODD polypeptide in a samplecomprising the steps of:(a) contacting a sample with an antibodyaccording to claim 6, and (b) detecting specific binding of the antibodyto the SODD polypeptide as an indication of the presence of the SODDpolypeptide in the sample.
 18. A method for detecting a SODD polypeptidein a sample comprising the steps of:(a) contacting a sample with anantibody according to claim 7, and (b) detecting specific binding of theantibody to the SODD polypeptide as an indication of the presence of theSODD polypeptide in the sample.
 19. A method for detecting a SODDpolypeptide in a sample comprising the steps of:(a) contacting a samplewith an antibody according to claim 8, and (b) detecting specificbinding of the antibody to the SODD polypeptide as an indication of thepresence of the SODD polypeptide in the sample.
 20. A method fordetecting a SODD polypeptide in a sample comprising the steps of:(a)contacting a sample with an antibody according to claim 9, and (b)detecting specific binding of the antibody to the SODD polypeptide as anindication of the presence of the SODD polypeptide in the sample.
 21. Amethod for detecting a SODD polypeptide in a sample comprising the stepsof:(a) contacting a sample with an antibody according to claim 10, and(b) detecting specific binding of the antibody to the SODD polypeptideas an indication of the presence of the SODD polypeptide in the sample.22. A method for detecting a SODD polypeptide in a sample comprising thesteps of:(a) contacting a sample with an antibody according to claim 11,and (b) detecting specific binding of the antibody to the SODDpolypeptide as an indication of the presence of the SODD polypeptide inthe sample.